Variola (smallpox) remains a bioterrorism threat. The Dryvax(TM) vaccine has toxicities, especially in immune compromised persons. Little is known about the specific CD8 T cell response induced by intradermal scarification with Dryvax. We propose detailed studies, using a proven, and somewhat unique, technology, of the vaccinia epitopes recognized by CD8 T-cells, and studies of the homing receptors expressed by CD8 T-cells. The new information should be applicable to immunogenicity evaluations of newer candidate smallpox candidate vaccines. We propose an antigen discovery approach interrogating libraries of cloned vaccinia genomic DNA with vaccinia-specific human CD8 CTL clones from recent vaccinees. Dryvax programs expression of a skin-homing adhesion molecule, CLA, on virus-specific CD4 T-cells. Similar homing research is proposed for vaccinia-specific CD8 T-cells. T-cell homing could be relevant to the pathogenesis of Dryvax-associated eczema vaccinatum, which is seen in persons with atopic dermatitis, a condition marked by excess CLA expression. Aim 1 will define the immunodominant antigens and epitopes of vaccinia recognized by CD8 CTL restricted by prevalent HLA alleles. We hypothesize that responses to immunodominant CTL epitopes will be present at high magnitude shortly after vaccination. To test this, SubAims are 1.1) Derive vaccinia-specific CD8 CTL clones from recent vaccinees, 1.2) Determine their fine specificity, and 1.3) Measure the magnitude of responses to immunodominant epitopes. Aim 2 will measure the expression and function of skin-homing-associated adhesion and chemokine receptor molecules on the surface of vaccinia-reactive CD8 T-cells. We hypothesize that vaccinia-specific CD8 T-cells will specifically express CLA, CCR4, and CCR10. Subaims will be 2.1) Measure CLA, CCR4, and CCR10 expression on vaccinia-specific CD8 cells, 2.2) Measure E-selectin binding by vaccinia-specific CD8 cells, and 2.3) Compare CLA and E-selectin binding by vaccinia-specific T-cells in recent and distant vaccines.